ParAB are essential in H. neptunium

To further investigate the role of ParAB in chromosome segregation in H. neptunium, we intended to gen-erate single in-frame deletions by double homologous recombination. However, we did not succeed in deleting either parA or parB, which suggests that they are essential for viability in H. neptunium.

Therefore, we attempted to create conditional mutants by introducing a fluorescently tagged copy of the respective gene at the locus of the zinc-inducible promoter and deleting the native copy at the endoge-nous locus. For ParB, no conditional mutant could be generated, since double homologous recombination gave exclusively rise to the wild-type genotype (data not shown). In the case of ParA, we were able to generate a conditional mutant. Microscopic analysis revealed that ParA depletion leads to elongated and misshaped cells with elongated stalks and bud compartments, as corroborated by flow cytometry analysis (see Figure 2-6 B & D and Figure S6-1 A). However, there was also a subpopulation of cells that showed wild-type morphology and cell size (see Figure 2-6 B & D). It should be noted that cells were also able to grow in the absence of inducer due to leaky expression of parA-venus from the zinc promoter (see Figure 2-6 C). Hence, long depletion times were required to detect morphological changes. Furthermore, this basal ParA level in the absence of induction might also be responsible for the fraction of cells showing wild-type morphology upon ParA depletion. Production of ParA-Venus from the zinc-inducible promoter mostly complemented the ParA depletion phenotype, since the majority of cells showed wild-type mor-phology. Nonetheless, the inducible ParA-Venus fusion did not lead to a complete complementation of the phenotype (see Figure 2-6 B & D and Figure S6-1 A). This might be caused by the altered ParA ex-pression level (inducible instead of native promoter) or the ParA-Venus fusion might not be fully func-tional.

Figure 2-5: Localization of ParB-YFP in the absence of BacAB. A) Cells of strain AJ74 (ΔbacAB parB::parB-yfp) were grown in MB to exponential phase, and visualized by DIC and fluorescence microscopy B) For time-lapse microscopy, cells of H. neptunium AJ74 were grown in MB medium to exponential phase, transferred to an MB agarose pad, and visualized at 30 min intervals by DIC and fluorescence microscopy. Overlays of DIC and fluorescence micrographs are shown. Bar 3 µm.

In the next step, we wanted to assess if chromosome segregation was hindered upon ParA depletion.

Demographic representation of nucleoid localization in budding cells revealed that DNA segregation was impaired in the vast majority of cells, since no DNA could be detected in the buds (see Figure 2-7). By comparison, DNA was segregated to the bud compartment in the majority of cells in the wild type con-trol (see Figure 2-7). Additionally, flow cytometry analysis of DNA content revealed that many cells had more than one or two chromosomes upon ParA depletion, while wild-type cells contained either one or two chromosomes (see Figure 2-6 E). This indicates that chromosome replication still takes place but, due to impaired segregation, DNA accumulates in the mother cell. However, there was also a fraction of cells, in which DNA segregation occurred normally (see Figure 2-7 B, left panel). This is most likely due to the basal ParA level present in the cells (see above). Furthermore, we could also observe the presence of anu-cleate cells (see Figure 2-7 A, asterisks & Table 6-5). In most cases, these cells did not have the shape of normal swarmer cells that usually bud off upon cell division, but rather looked like thickened parts of a stalk. This suggests that cell division takes place at low frequency even without chromosome segregation

Figure 2-6: Effects of ParA depletion on cell morphology, cell size, and DNA content. A) Construction of strain AJ46. A copy of parA-venus was introduced at the site of the zinc-inducible promoter and parA was deleted from its native locus.

B) Effects of ParA depletion on cell morphology. Cells of strain AJ46 (ΔparA PZn::PZn-parA-venus) were grown in the presence of inducer (0.3 mM ZnSO4), washed, shifted to ZnSO4-free medium, and grown for 45 h. After 16.5 h and 41 h of depletion, cells were diluted 1:10 and 1:3, respectively, in MB medium. For reinduction of ParA, cells were shifted to MB medium supple-mented with 0.5 mM ZnSO4. Morphological changes were assessed by DIC microscopy. Bar 5 µm. C) Immunoblot analyses of ParA-Venus levels of samples described in (B). D) & E) Effects of ParA depletion on cell size and chromosome content. Cells of strain AJ46 were grown in MB medium in the presence or absence of 0.3 mM ZnSO4 (inducer). WT: H. neptunium wild type was grown in MB. Subsequently, exponentially growing cells were analyzed by flow cytometry. To analyze the DNA content by flow cytometry, cells were stained with Vybrant® DyeCycle™ Orange for 25 min beforehand.

taking place beforehand. However, the presence of elongated cells indicates that cell division appears to be impaired. As mentioned above, the induction of ParA-Venus from the zinc-inducible promoter did not complement the depletion phenotype completely. Consistently, DAPI staining revealed that chromosome segregation was impaired even upon induction of ParA-Venus in a subpopulation of cells (see Figure 2-7 B, middle panel).

In the next step, we intended to analyze the localization of the origin region in the absence of ParA.

However, we did not succeed in introducing a fluorescently tagged version of ParB in the ParA depletion strain. Therefore, and due to the need for very long depletion times to detect morphological changes, we were seeking for an alternative approach to impair ParA functioning.

In C. crescentus, a missense mutation in the ATPase domain (K20R) of ParA leads to a dominant negative effect when produced in addition to wild-type ParA, resulting in incomplete chromosome segregation (68). We therefore mutated the H. neptunium ParA accordingly (see Figure 2-8 A) and introduced the mu-tated, fluorescently tagged version under the control of the copper-inducible promoter. To visualize the localization of the origin region, ParB was fluorescently tagged with Cerulean in the same strain. In order to analyze the effects of the ParA mutation on cell morphology and origin localization, we performed time-course microscopy. Before induction of the mutated ParA (ParA K18R), the cells already showed morphological alterations, most likely due to the low basal activity of the copper-inducible promoter (data not shown). Nevertheless, many cells also showed wild-type morphology and origin segregation. After 4 and 6 h of induction of the mutated ParA, cells showed an aberrant morphology with elongated stalks and buds (see Figure 2-8 B and Figure S6-1 B). It appeared that although cell division was impaired, the

Figure 2-7: Effects of ParA depletion on chromosome segregation. A) Cells of strain AJ46 were depleted of ParA for 45h, subsequently stained with DAPI for 20 min, and visualized by DIC and fluorescence microscopy. Asterisks indicate DNA-free cells. Bar 5 µm. B) Demographic representation of the DAPI (DNA) signal in budding cells of strain AJ46 grown in the pres-ence or abspres-ence of inducer (0.3 mM ZnSO4) and in H. neptunium wild-type cells. Demographs are aligned with the mother cell on the right and the bud compartment on the left. Cells of strain AJ46 were grown in MB with or without 0.3 mM ZnSO4 and H. neptunium wild-type cells were grown in MB to exponential phase, stained with DAPI for 20 min, and visualized by DIC and fluorescence microscopy.

budding process did not stop and that the buds somehow grew further into the stalk, resulting in a clubbed morphology (see Figure 2-8 B). Moreover, origin segregation was impaired in these cells, since there was hardly any ParB-Cerulean focus detected in the bud compartment (see Figure 2-8 B). These observations were also confirmed by flow cytometry analysis. Here, many cells had an increased cell size after 4 h of induction of the mutated ParA compared to the wild type, the non-induced control or a con-trol strain carrying a fluorescently tagged, wild-type ParA at the locus of the copper-inducible promoter (see Figure 2-9 C). When the induction time was prolonged, these defects became even more pronounced, resulting in elongated and misshaped cells with multiple ParB foci in the mother cell (see Figure 2-8 B).

This phenotype resembles the before mentioned ParA depletion phenotype.

Additionally, microscopic analysis of DAPI stained cells confirmed that no DNA is transported through the stalk to the bud compartment, whereas in the control strains DNA was detected in the bud (see Figure 2-9 A & B). Flow cytometry analysis further revealed that cells contained two or more chromosomes, while wild-type cells had either one or two chromosomes (see Figure 2-9 C). Moreover, as already seen for the conditional ParA mutant, anucleate cells were observed (see Figure 2-8 B & Figure 2-9 A, white aster-isks and Table 6-5).

Overall, we can conclude that the ParAB/parS system mediates chromosome segregation in the mother cell, since origin segregation, and DNA segregation in general, are incomplete when ParA functioning is impaired. Furthermore, one could speculate that completion of origin segregation in the mother cell might be required for the initiation of the second step (transport through stalk), indicating that origin segregation is a sequential process. Admittedly, we cannot exclude the formal possibility that ParA itself is required for origin segregation through the stalk. However, this seems rather unlikely, since the generic

Figure 2-8: Effects of ParA K18R production on cell morphology and origin segregation. A) Alignment of the N-terminal part of the primary sequence of ParA of C. crescentus NA1000 and H. neptunium ATCC 15444. The Walker A motif is highlighted in light grey. The conserved lysine that was previously mutated in C. crescentus (68) and mutated in this work in H. neptunium is highlighted in purple. B) Effects of ParA K18R production on cell morphology and origin segregation. Cells of strain AJ79 (parB-cerulean PCu::PCu-parA(K18R)-venus) were grown in MB medium to exponential phase, induced with 0.5 mM CuSO4, and growth was continued for 18.5 h. Samples were taken at the indicated time points. Morphological changes and ParB localization were analyzed by DIC and fluorescence microscopy. Asterisks indicate cells without a ParB-Cerulean signal. Bar 5 µm. C) ParA K18R-Venus and ParB-Cerulean levels in cells grown as described in (A) were analyzed by immunoblot analysis using an GFP anti-body.

ParABS system requires non-specific chromosomal DNA for proper functioning (65, 77) and the stalk appears to be free of DNA before it is traversed by the ParB/parS complex. Furthermore, impairment of ParA activity resulted in amorphous and elongated cells, suggesting that chromosome segregation might be coupled, to some extent, to other cell cycle events such as budding and/or cell division.

Figure 2-9: Effects of ParA K18R production on cell size and DNA content. A) Effects of ParA K18R production on chromosome segregation. Cells of strain AJ79 (parB-cerulean PCu::PCu-parA(K18R)-venus) and AJ80 (parB-cerulean PCu::PCu -parA-venus) were grown in MB medium to exponential phase, induced with 0.3 mM CuSO4 for 4.5 h, subsequently stained with DAPI for 25 min, and visualized by DIC and fluorescence microscopy. Additionally, cells of strain AJ79 were grown in MB to exponen-tial phase, but growth was continued in MB without inducer for 4.5 h and samples were analyzed accordingly. Asterisks indicate DNA-free cells. Bar 5 µm. B) Demographic representation of DAPI signal and ParB-Cerulean localization in budding cells of strains described in (A). Demographs are aligned with the mother cell on the right and the bud compartment on the left. C) Effects of ParA K18R production on cell size and chromosome content. Cells of strain AJ79 and AJ80 were grown in MB medi-um to exponential phase and induced with 0.3 mM CuSO4 for 4.5 h. Additionally, cells of strain AJ79 and H. neptunium wild type were grown in MB to exponential phase but growth was continued in MB without inducer for 4.5 h. Subsequently, cells were analyzed by flow cytometry. To analyze the DNA content by flow cytometry, cells were stained with Vybrant® DyeCycle™

Orange for 25 min.